Autoimmune disease model animal

ABSTRACT

Pemphigus vulgaris (PV) is an autoimmune disease with a possible fatality of the skin and mucosae which is induced by an antibody against desmoglein 3 (Dsg3). Persistent production of anti-Dsg3 IgG can be induced by adoptively transferring spleen cells of a DSG3−/− mouse immunized with rDsg3 into an RAG2−/− immunodeficient mouse expressing Dsg3 protein. This IgG in the blood binds to the Dsg3 protein in vivo, induces the breakage of intercellular adhesion of keratinocytes and thus brings about the phenotype of pemphigus vulgaris involving the formation of blisters in the oral mucosa and the disappearance of resting hair. These effects are sustained over 6 months. By using this method, active disease model animals relating to various autoimmune diseases can be constructed.

The present application is a continuation of U.S. patent applicationSer. No. 09/937,739, which claims the benefit of PCT InternationalApplication No. PCT/JP00/02023, filed Mar. 30, 2000, which claimsbenefit of Japanese Patent Application No. 11/91408, filed Mar. 31,1999, which are hereby incorporated in their entirety.

TECHNICAL FIELD

The present invention relates to autoimmune disease model animals andmethods for producing them.

BACKGROUND ART

Pemphigus vulgaris (PV) is an autoimmune disease with involvement ofskin and mucous membrane blistering, which is sometimes fatal, and ishistologically characterized by blistering in the epidermis as well asimmunopathologically characterized by the presence of autoantibody IgGto the cell surface of keratinocyte (Stanley, J. R. Pemphigus. InDermatology in General Medicine. I. M. Freedberg, A. Z. Eisen, K. Wolff,K. F. Austen, L. A. Goldsmith, S. I. Katz, and T. B. Fitzpatrick, eds.McGraw-Hill, New York, 654-666 (1998)). Patients with pemphigus vulgarisclinically manifest diffuse flaccid blister and erosion. These can beformed in all the stratified squamous epithelia. Without appropriatetherapy, the widespread lesions on the skin result in the leakage ofbody fluid or secondary bacterial infection, and as a result pemphigusvulgaris may be fatal. The prognosis of pemphigus can be improved bysystemic administration of corticosteroid and immunosuppression therapy,but the mortality remains considerably high because of death due tocomplications caused by the therapy.

The target antigen for pemphigus vulgaris was first identified as a 130kD glycoprotein through immunoprecipitation of keratinocyte extract(Stanley, J. R. et al., J. Clin. Invest. 70:281-288 (1982); Stanley, J.R. et al., J. Clin. Invest. 74:313-320 (1984)). Then, cDNA for thepemphigus vulgaris antigen was isolated via immuno-screening of a humankeratinocyte expression library using affinity-purified autoantibodyspecific to the pemphigus vulgaris antigen (Amagai, M. et al., Cell67:869-877 (1991)). Nucleotide sequence analysis has revealed that thepemphigus vulgaris antigen belongs to the superfamily of genes forcadherins that are intercellular adhesion molecules. The pemphigusvulgaris antigen is a membrane protein located in desmosome (Karpati, S.et al., J. Cell Biol. 122:409-415 (1993)), and it was named desmoglein 3(Dsg3) (Amagai, M. Adv. Dermatol. 11:319-352 (1996)).

There is much evidence showing that autoantibody IgG against Dsg3protein plays a pathogenic role in pemphigus vulgaris. Firstly, it hasbeen reported that activity of the disease correlates to the antibodytiter in blood over time by indirect fluorescent antibody technique(Sams Jr, W. M. & Jordon, R. E., Br. J. Dermatol. 84:7-13 (1971)) orELISA (Ishii, K., et al., J. Immunol. 159:2010-2017 (1997); Amagai, M.,et al., Br. J. Dermatol. 140:351-357 (1999)). Secondly, a newborn fromthe mother affected with pemphigus vulgaris is also transiently affectedwith the disease due to the IgG transferred across the placenta from themother (Merlob, P. et al., Pediatrics 78:1102-1105 (1986)). As the IgGderived from the mother is catabolized, the symptom is remitted.Thirdly, the IgG derived from patients with pemphigus vulgaris caninduce blistering in tissue-cultured skin in the absence of complementand inflammatory cell (Schiltz, J. R., & Michel, B., J. Invest.Dermatol. 67:254-260 (1976); Hashimoto, K. et al., J. Exp. Med.157:259-272 (1983)). Fourthly, passive transfer of the IgG derived fromsera of patients into newborn mice causes intraepidermal blisterformation with typical histological characteristics (Anhalt, G. J. etal., N. Engl. J. Med. 306:1189-1196 (1982)). Fifthly, depletion ofpatient-derived serum by immuno-absorption using recombinant Dsg3protein (rDsg3) comprising extracellular domain thereof removespathogenicity of the serum and inhibits blistering in newborn mice(Amagai, M. et al., J. Clin. Invest. 94:59-67 (1994)). Finally, antibodyaffinity-purified with rDsg3 exhibits pathogenicity and thus results inthe formation of blister with histological characteristics of pemphigusvulgaris in newborn mice (Amagai, M. et al., J. Clin. Invest. 90:919-926(1992); Amagai, M. et al., J. Clin. Invest. 102:775-782 (1998)).

Based on these studies, pemphigus vulgaris is one of thebest-characterized autoimmune diseases with respect to the processesafter the generation of autoantibody in particular. Thus pemphigusvulgaris is now an excellent disease model for tissue-specificautoimmune diseases to study cellular mechanisms underlying theproduction of autoantibody or destruction of self-tolerance, as well asto develop therapeutic methods specific to the diseases. As the firststep toward the goals, it is demanded to develop active disease animalmodel for pemphigus vulgaris.

Most of experimental autoimmune disease animal models are provided byrepeated injection of autoantigen with a variety of adjuvants. However,as exemplified by the case of myasthenia gravis, in which the frequencyof generation of the active disease in mice immunized with acetylcholinereceptor (T. californica) varies considerably depending on the strains,the success of this method is thus highly empirical (Berman, P. W. etal., Ann. N.Y. Acad. Sci. 377:237-57 (1981)).

Previously, an in vivo experimental model for pemphigus vulgaris wasdeveloped by the reconstruction of severe combined immunodeficiency(SCID) in mice using PBMC derived from patients with pemphigus vulgaris(Juhasz, I. et al., J. Clin. Invest. 92:2401-7 (1993)). With this model,lymphocytes from the patients produced circulating autoantibody at a lowtiter, but it was rare that active intraepidermal blistering withdeposition of human IgG was found in mouse skin. When human skin wastransplanted on SCID mouse, blisters similar to those in pemphigusvulgaris were recognized on the transplanted skin. However, it cannot bedenied that the cause of blister formation in this model is aninflammatory response due to the tissue incompatibility with human PBMCand skin. Thus there was no established active disease model forpemphigus vulgaris.

DISCLOSURE OF THE INVENTION

The present invention provides autoimmune disease model animals and amethod for producing them. More specifically, the present inventionprovides non-human mammals showing phenotypes of the autoimmune diseasein which activation of T cells and B cells reactive to the antigenprotein for the autoimmune disease followed by stable production ofautoantibody are induced and provides a method for producing them. In apreferable embodiment, the model animal can be provided by thetransplantation of immune cells including B cells producing antibodyagainst the antigen protein of the autoimmune disease and/or T cellsthat are reactive to the antigen protein.

To achieve the above described objective, first, the present inventorsaimed at the production of autoantibody in mice by employing thepreviously used typical method with repeated injection. Specifically,three strains of mice, BALB/c(H-2^(d)), C3H/HeJ(H-2^(k)), andC57BL/6N(H-2^(b)) were immunized with human or mouse Dsg3 protein.Complete Freund's adjuvant was used in the primary immunization, andthen booster immunization was carried out 3 or 7 times by usingincomplete Freund's adjuvant. However, with this method, no miceproduced antibody capable of reacting to mouse Dsg3 protein (Table 1)and showed phenotype of pemphigus vulgaris at all.

Based on this result, the present inventors set up the hypothesis thatself-tolerance to Dsg3 protein prevents the production of pathogenicantibody in mouse body. According to the hypothesis, it can be assumedthat the immune system is not exposed to Dsg3 protein during thedevelopmental stages in Dsg3-deficient mouse created by gene-targetingtechnique and thus the mouse does not acquire self-tolerance to Dsg3protein.

In order to demonstrate the hypothesis, the present inventors studiedwhether it was possible for Dsg3-deficient mouse immunized with Dsg3protein to produce antibody against Dsg3 protein. From the result, itwas revealed that when immunized with Dsg3 protein a homozygous DSG3gene-deficient DSG3−/− mouse much more efficiently produced the antibodythan a heterozygous DSG3 gene-deficient DSG3+/− mouse (FIG. 1A). Inaddition, the antibody produced by DSG3−/− mouse was capable of bindingto mouse Dsg3 protein on the keratinocyte, but the antibody from DSG3+/−mouse was not (FIG. 1B). Specifically, it was revealed thatself-tolerance to DSG3 protein had not been established in DSG3−/− mouseand the produced antibody recognized mouse Dsg3 protein as an antigen.

Thus, the present inventors next aimed at the production of antibodyagainst Dsg3 protein and expression of phenotype of pemphigus vulgarisin RAG2−/− immunodeficiency mouse by collecting splenocytes (which havecapability of producing antibody against DSG3 protein) from DSG3−/−mouse immunized with Dsg3 protein and adoptively transferring them intothe immunodeficiency mouse. Such RAG2−/− mice express Dsg3 protein, butthe mice have neither mature T cells nor B cells because they aredeficient in rearrangement of T cell receptor genes and immunoglobulingenes (namely, they are immunodeficient).

As a result, in RAG2−/− mice in which splenocytes from DSG3−/− mouse hadbeen transplanted, the encounter of Dsg3 protein-specific lymphocytesamong splenocytes with endogenous Dsg3 protein resulted in permanentproduction of the antibody against Dsg3 protein (FIG. 2A). In addition,it was found that RAG2−/− mice having the immunized DSG3−/− splenocytesshowed nearly identical phenotype of DSG3−/− mouse (Koch, P. J., et al.,J. Cell Sci. 111:2529-2537 (1998); Koch, P. J., et al., J. Cell Biol.137:1091-1102 (1997)). All of the mice exhibited erosive lesions inmucous membranes with epidermal separation just above the basal celllayer and telogen hair loss (FIG. 3). The presence of nearly identicalphenotype reproduced by adoptive transfer of DSG3−/− splenocytes inRAG2−/− recipient mice demonstrated that the produced antibody wasspecific and pathogenic.

The specificity of the antibody can also be verified by the fact thatthe in vivo deposition is not detectable in other simple epitheliaexpressing Dsg2 protein (Schafer, S. et al., Exp. Cell Res. 211:391-9(1994)) and upper part of epidermis expressing Dsg1 protein (FIG. 3G)(Amagai, M. et al., J. Invest. Dermatol. 106:351-355 (1996)).

Thus, the present invention provides the first disease mouse model forpemphigus and a method for producing them. The method of the presentinvention, because of the nature thereof, can be widely applicable tothe preparation of model animals for other autoimmune diseases in whichassociated autoimmune targets have been identified.

Accordingly the present invention relates to autoimmune disease modelanimals and a method for producing them, more specifically relates to:

(1) a non-human mammal showing a phenotype of autoimmune disease throughproduction of an antibody reacting to an antigen protein for anautoimmune disease or T cell activation;

(2) the non-human mammal of (1), wherein immune cells from a non-humanmammal lacking an antigen gene for the autoimmune disease have beentransplanted to the non-human mammal;

(3) the non-human mammal of (1), wherein immune cells from a non-humanmammal that lacks the antigen gene for the autoimmune disease and thathas been immunized with the antigen protein have been transplanted tothe non-human mammal;

(4) the non-human mammal of (2) or (3), wherein the immune cells aretransplanted to an immunodeficient non-human mammal;

(5) the non-human mammal of (4), wherein the immunodeficient non-humanmammal is a non-human mammal that lacks the RAG2 gene;

(6) the non-human mammal of any one of (2) to (5), wherein the immunecells are splenocytes;

(7) the non-human mammal of any one of (1) to (6), wherein theautoimmune disease is pemphigus vulgaris;

(8) the non-human mammal of (7), wherein the antigen protein isdesmoglein 3 protein;

(9) the non-human mammal of any one of (1) to (8), wherein the non-humanmammal is a rodent;

(10) the non-human mammal of (9), wherein the rodent is a mouse;

(11) a method for producing a non-human mammal showing a phenotype ofautoimmune disease through production of an antibody reacting to anantigen protein for an autoimmune disease or T cell activation, whichcomprises the steps of:

-   -   (a) immunizing, with the antigen protein for the autoimmune        disease, a non-human mammal that lacks the antigen gene for the        autoimmune disease,    -   (b) preparing immune cells from the non-human mammal, and    -   (c) transplanting the immune cells to a non-human mammal having        the antigen protein;

(12) the method of (11), wherein the immune cells are transplanted to animmunodeficient non-human mammal;

(13) the method of (12), wherein the immunodeficient non-human mammal isa non-human mammal that lacks the RAG2 gene;

(14) the method of any one of (11) to (13), wherein the immune cells aresplenocytes;

(15) the method of any one of (11) to (14), wherein the autoimmunedisease is pemphigus vulgaris;

(16) the method of (15), wherein the antigen protein is desmoglein 3protein;

(17) the method of any one of (11) to (16), wherein the non-human mammalis a rodent; and

(18) the method of (17), wherein the rodent is a mouse.

The model animal of the present invention can show phenotype ofautoimmune disease through the stable production of antibody reacting tothe antigen protein for the autoimmune disease or sustained activationof T cell.

There is no particular restriction on the type of objective disease forwhich model animals are to be prepared in accordance with the presentinvention, as far as the disease is an autoimmune disease. Suchautoimmune diseases include, for example, but not limited to, pemphigusvulgaris, myasthenia gravis, autoimmune hemolytic anemia, Basedow'sdisease, Hashimoto's disease, Goodpasture's syndrome, autoimmunediabetes mellitus, multiple sclerosis, etc.

Animals to be utilized for creating the model animal are preferablynon-human mammals. There is no restriction on such non-human mammals, asfar as gene-disrupted animals can be created from them. Preferableanimals include rodents, e.g., mouse.

The model animals in accordance with the present invention can becreated by immunizing antigen gene-deficient non-human mammals with theantigen protein for the autoimmune disease, removing the immune cellsthereof, and then transplanting the cells to other non-human mammalshaving the antigen protein.

Animals having the disputed antigen gene can be created by a methodknown to those skilled in the art. The antigen gene to be disruptedincludes, for example, but not limited to, the DSG3 gene when theautoimmune disease is pemphigus vulgaris; the acetylcholine receptorgene for myasthenia gravis; the TSH receptor gene for Basedow's diseaseor Hashimoto's disease; the type IV collagen gene for Goodpasture'ssyndrome; the myelin basic protein gene for multiple sclerosis, etc.

Further, immune cells can be obtained from the thymus, lymph node,spleen, liver, intestinal epithelium, peripheral blood, etc. but are notlimited to those from the tissues. The spleen abundantly contains matureimmune cells and thus is a preferable organ for the immune cells. It ispreferable that the animal (donor) from which immune cells are preparedand the animal (recipient) to which lymphocytes derived from the immunecells are transferred belong to a same species and have a same geneticbackground thereby preventing the onset of GVHD which may cause tissuedestruction in the recipient.

In addition to this, it is preferable that the recipient hasimmunodeficiency thereby preventing the rejection of lymphocytes derivedfrom immune cells transferred. For example, SCID mouse, nude mouse aswell as an animal of which RAG2 gene has been disrupted may be used asthe immunodeficient animal. Furthermore, MHC-knockout mouse or common ychain-knockout mouse can also be used but it is not limited thereto.

The immunization with the antigen protein from the donor, preparation ofimmune cells from the donor, and transplantation of the immune cells tothe recipient can be carried out, for example, by the methods asdescribed in the Examples.

The model animal created in accordance with the present invention canshow phenotype of autoimmune disease through the stable production ofantibody reacting to the antigen protein for the autoimmune disease orsustained activation of T cell. In the model animal for pemphigusvulgaris, major phenotype includes weight loss and reversible telogenhair loss. Further, among autoimmune diseases other than pemphigusvulgaris, phenotype may include reduced muscle power in myastheniagravis; anemia in autoimmune hemolytic anemia; hyperthyroidism inBasedow's disease; hypothyroidism in Hashimoto's disease; nephropathyand pulmonary disorders in Goodpasture's syndrome; glucosuria inautoimmune diabetes mellitus; and neuroparalysis in multiple sclerosis.

One can use these model animals for developing therapeutic agents ormethods for the diseases, administering to them test compounds ofinterest for therapeutic effects on autoimmune diseases and observingphenotypes thereof. Particularly the major phenotype includes weightloss and reversible telogen hair loss in pemphigus vulgaris model mouseprepared in accordance with the present Example and the phenotypes lastover 6 months, and therefore it is possible to readily and objectivelyevaluate the effectiveness of each therapeutic agent or method based onthe observation without sacrificing the mouse. In addition, these modelmice are very useful for clarifying cellular mechanism underlying theproduction of antibody against the antigen protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram (A) and photograph (B) displaying the productionof anti-Dsg3 IgG that is capable of in vivo binding in DSG3−/− mouse.(A): DSG3−/− mouse and the +/− littermate mice thereof were immunizedwith mouse rDsg3, and then the titers of anti-Dsg3 IgG were measuredover time by ELISA. An arrow indicates the primary immunization withmouse rDsg3 using complete Freund's adjuvant, and an arrow headindicates booster immunization with mouse rDsg3 using incompleteFreund's adjuvant. DSG3−/− mouse efficiently produced much moreanti-Dsg3 IgG than the +/− littermate mice. (B): while intercellularjunctions of cultured keratinocytes were stained with the serum derivedfrom the immunized DSG3−/− mouse (a), they were not stained with theserum derived from the +/− littermate mice (b). Bar represents 50 μm.

FIG. 2 shows the production of anti-Dsg3 IgG in recipient RAG2−/− miceafter the transfer of the immunized DSG3−/− splenocytes. (A): ELISAscores to mouse rDsg3 were obtained with RAG2−/− mouse that had receivedsplenocytes from the immunized DSG3−/− mouse [RAG2−/− (DSG3−/−)] orRAG2−/− mouse that had received splenocytes from the immunized DSG3+/−mouse [RAG2−/− (DSG3+/−)]. The RAG2−/− mouse having DSG3−/− splenocyteshad the sustained production of anti-Dsg3 IgG. An arrow indicates thefirst day of telogen hair loss phenotype. In contrast, ELISA for RAG2−/−mouse having DSG3+/− splenocytes was always negative over time. (B): thetime course of varying body weight of recipient RAG2−/− mouse wasplotted. After 10 to 14 days, the increase in weight delayed in RAG2−/−mice having DSG3−/− splenocytes as compared with mice having DSG3+/−splenocytes, and then the weight continued to decrease. Several micedied (\), but the remaining several mice survived and then their weightsincreased.

FIG. 3 is a photograph showing the expression of pemphigus vulgarisphenotype in RAG2−/− mice in which the immunized DSG3−/− splenocyteshave been transferred. Around 7 to 14 days after the transfer ofsplenocytes, weight loss was recognized in RAG2−/− mice having DSG3−/−splenocytes (A, bottom) when compared with mice having DSG3+/−splenocytes (A, top). Several mice showed the onset of erosion with scabaround noses and cheeks where they scratched (B). Based on histologicaldiagnosis of RAG2−/− mouse having DSG3−/− splenocytes, intraepidermalblister formation was found immediately above the basal layer of mucosalepithelium (C, hard palate; D, upper part of the esophagus).Inflammatory infiltrates were recognized below the erosion foci (E,upper part of the esophagus). In vivo IgG deposition was recognized oncellular surface of keratinocyte in mucosal epithelium by directimmunofluorescence method (F, hard palate) and skin (G, around nose)(white part of the diagram). Bar represents 50 μm.

FIG. 4 is a photograph showing a telogen hair loss phenotype of RAG2−/−mouse, in which the immunized DSG3−/− splenocytes had been transferred,similar to that observed in DSG3−/− mouse. About 15 to 25 days after thetransfer, RAG2−/− mouse having DSG3−/− splenocytes showed partial hairloss (A, B). In hair-pull test with adhesive tape, a bunch of hairs wereadhered on the tape in the case of RAG2−/− mouse having DSG3−/−splenocytes (C, left), but no hair was adhered in the case of RAG2−/−mouse having DSG3+/− splenocytes (C, right). After that, a mosaic of newhair was recovered in the area without hair (D, arrow). Histologicaldiagnosis showed the presence of acantholysis between cells of hair bulband basal layer of outer root sheath epithelium (E, arrow) as well asthe presence of empty expanded telogen hair follicle (F, arrow). Bydirect immunofluorescence method, in vivo IgG deposition was found onthe cellular surface of keratinocyte in the hair root (G, H) (white partof the diagram). Bar represents 50 μm.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is illustrated in detail below with reference toExamples, but is not to be construed as being limited thereto.

EXAMPLE 1 Production of Recombinant Mouse Dsg3 Protein

A cDNA encoding the entire extracellular domain of mouse Dsg3 (GenbankU86016) was amplified by PCR using appropriate primers(5′-CCGAGATCTCCTATAAATATGACCTGCCTCTTCCCTAGA-3′/SEQ ID NO: 1,5′-CGGGTCGACCCTCCAGGATGACTCCCCATA-3′/SEQ ID NO: 2) and using a phageclone containing mouse Dsg3 cDNA (a gift from Dr. Jouni Uitto) as atemplate; amplified fragment was subcloned (pEVmod-mDsg3-His) byreplacing it with human Dsg3 cDNA in pEVmod-Dsg3-His vector (Ishii, K.,et al., J. Immunol. 159:2010-2017 (1997)). A recombinant baculo-protein,mouse rDsg3, was prepared as described previously (Amagai, M. et al., J.Clin. Invest. 94:59-67 (1994); Amagai, M. et al., J. Invest. Dermatol.104:895-901 (1995)).

EXAMPLE 2 Immunization of Wild-Type DSG3+/+ Mouse with Mouse Dsg3Protein

First, attempts were made to produce antibodies against Dsg3 protein ina variety of wild-type mouse strains after immunizing with human ormouse rDsg3 (Table 1).

Mice were sensitized with 5 μg of purified mouse or human rDsg3 byintraperitoneal injection with complete Freund's adjuvant (CFA). Thenbooster immunization was carried out every week, 3 or 7 times, withmouse or human rDsg3 using incomplete Freund's adjuvant (IFA). An ELISAtest for antibody production was conducted 3 days after each boosterimmunization.

In ELISA assay for blood IgG against mouse Dsg3 protein (mDsg3) or humanDsg3 protein (hDsg3) in mice, mouse or human rDsg3 was used as a coatingantigen. More specifically, a 96-well microtiter plate was coated with100 μl of 5 μg/ml purified mouse or human rDsg3 at 4° C. overnight. Allserum samples were diluted 50 to 5,000 times and then incubated on a96-well ELISA plate at room temperature for 1 hour. After the sampleswere incubated with peroxidase-conjugated goat anti-mouse IgG antibody(MBL, Nagoya, Japan) at room temperature for 1 hour, the coloringreaction was carried out by using 1 mM tetramethylbenzidine as asubstrate for peroxidase (Ishii K et al., J Immunol 159: 2010-2017,1997; Amagai M et al., Br J Dermatol 140:351-357, 1999). The respectivesamples were analyzed in duplicate. A single serum sample obtained fromDSG3−/− mouse immunized with mouse rDsg3 was used as a positive controland serum derived from a non-immunized mouse was used as a negativecontrol. ELISA score was obtained as an exponent in a value calculatedby [(sample OD−negative control OD)/(positive control OD—negativecontrol OD)×100] (Table 1).

Further, the production of antibody against Dsg3 protein was tested byimmuno-fluorescent staining of cultured keratinocytes. Mousekeratinocytes from cell line PAM212 (Yuspa, S. H. et al., Cancer Res.40:4694-4703 (1980)) or human keratinocytes from cell line KU8(Tsukamoto, T., Keio J. Med. 38:277-293 (1989)) were incubated togetherwith mouse serum sample 20-fold diluted with DMEM containing 10% FCS at37° C. under humid air containing 5% CO₂ for 30 minutes. Subsequentlythe cells were washed with PBS(−) and then fixed with 100% methanol at−20° C. for 20 minutes; the cells were incubated with fluoresceinisothiocyanate (FITC)-labeled goat anti-mouse IgG antibody (DAKO,Copenhagen, Denmark) at room temperature for 30 minutes. The stain wasobserved with a fluorescence microscope (Nikon, Eclipse E800).

The ELISA and immuno-fluorescence staining revealed that C57BL/6N mouseor BALB/c mouse produced IgG capable of reacting to only human rDsg3 butnot to mouse rDsg3 when immunized with human rDsg3 first using completeFreund's adjuvant and then using incomplete Freund's adjuvant. MerelyC3H/HeJ mice produced IgG capable of weakly cross-reacting to mouserDsg3, when observed by ELISA. When these three strains of mice wereimmunized with mouse rDsg3, no mice from the three strains produced IgGrecognizing mouse or human rDsg3 in the evaluation by three methods(ELISA, indirect fluorescent antibody technique, and living keratinocytestaining). These findings suggested that wild-type DSG3+/+ mouse hadimmuno-tolerance to Dsg3 and thus it was difficult to allow for DSG3+/+wild-type mouse to produce antibody against mouse Dsg3. TABLE 1 Livingcell Mouse ELISA^(C) IIF^(d) staining^(e) strain n Antigen^(a) CFAIFA^(b) mDsg3 hDsg3 NMS NHS Pam KU8 C57BL/6N 2 hDsg3 1 3 18.4 93.4 −− +−− + BALB/c 3 hDsg3 1 3 6.3 85.4 −− + −− + C3H/HeJ 2 hDsg3 1 3 47.5167.7 −− + −− + C57BL/6N 3 mDsg3 1 7 10.6 10.1 −− −− −− −− BALB/c 3mDsg3 1 7 1.7 2.3 −− −− −− −− C3H/HeJ 3 mDsg3 1 7 5.4 7.5 −− −− ND ND

When recombinant human Dsg3 was used, “hDsg3” is provided in the columnof antigen in Table 1; when recombinant mouse Dsg3 was used, “mDsg3” isprovided (a). In the column of “CFA,” the number of immunizationtreatments with purified mouse or human recombinant Dsg3 using completeFreund's adjuvant (CFA) is indicated; in the column of “IFA,” the numberof booster immunizations conduced every week after the first one usingincomplete Freund's adjuvant (IFA) (3 or 7 times) is indicated (b).ELISA score was computed for human recombinant Dsg3 (hDsg3) or mouserecombinant Dsg3 (mDsg3) (c); if higher than 20.0, it can be judged aspositive. “IIF” of this Table indicates a result of indirectfluorescence antibody staining (IIF) of normal mouse skin (NMS) ornormal human skin (NHS) by using mouse serum (d). Further, “living cellstaining” of this Table indicates a result of living cell staining forcultured mouse keratinocytes from a cell line (Pam) or humankeratinocytes from a cell line (KU8) by using the mouse serum (e). “−”means negative; “+” means positive. “ND” indicates that the test was notdone.

EXAMPLE 3 Immunization of DSG3−/− Mouse and DSG3+/− Mouse with MouseDsg3 Protein

DSG3−/− mice were prepared by mating male DSG3−/− mice with femaleDSG3+/− mice (Koch, P. J., et al., J. Cell Biol. 137:1091-1102 (1997)).RAG2−/− mice, which had been obtained by back-crossing with B6.SJL-ptprcover 10 generations, were provided from Taconic (German Town, N.Y.)(Schulz, R.-J. et al., J. Immunol. 157:4379-4389 (1996)).

ELISA scores for mouse rDsg3 were determined after immunizing DSG3−/−mouse with mouse rDsg3 in order to verify the absence ofimmuno-tolerance to Dsg3 protein in DSG3−/− mouse.

Both DSG3−/− mice and DSG3+/− mice were sensitized with 5 μg of purifiedmouse rDsg3 by using complete Freund's adjuvant (0 day), and thenbooster was carried out with mouse rDsg3 by using incomplete Freund'sadjuvant after 8, 15, 22, and 28 days. The antibody production wastested by ELISA using mouse rDsg3 as a coating antigen in the samemanner as in Example 2.

The production of anti-Dsg3 IgG was found as early as 11^(th) day inDSG3−/− mice (n=4) and the titer continued to increase (FIG. 1A). WhenDSG3+/− mice were immunized repeatedly, the ELISA titer eventuallyincreased, but the titer was significantly lower than the titer forDSG3−/− mice observed on the 32^(nd) day (p<0.0001).

To determine whether the anti-Dsg3 IgGs produced by these mice can bindto Dsg3 protein on the keratinocytes in vivo, the same staining as inExample 2 was carried out by using mouse keratinocytes from a cell linePam212. When the serum derived from DSG3−/− mouse was added to culturemedia, the serum bound at intercellular adhesion sites of culturedkeratinocytes. However, no stain was detectable on the cell surface byusing sera derived from DSG3+/− mouse at all (FIG. 1B). There was no invivo IgG deposition in the epidermis of the immunized DSG3+/− mice. Thusthere is an extremely high possibility that the antibodies produced byDSG3+/− mouse are those against trace quantities of contaminants inpurified mouse rDsg3, against the C-terminal tag of mouse rDsg3, oragainst masked Dsg3 epitopes which are not accessible under the in vivocondition.

These results suggested that there was no immuno-tolerance to Dsg3 inDSG3−/− mouse and thus pathogenic IgG inhibiting Dsg3 function foradhesion was produced via immunizing DSG3−/− mouse with mouse rDsg3.

EXAMPLE 4 Permanent Production of Pathogenic Anti-Dsg3 IgG in RecipientRAG2−/− Mouse

Because DSG3−/− mouse has the deficient target antigen, it was predictedthat anti-Dsg3 IgG did not affect the phenotype in DSG3−/− mouse. Thus,an experiment was conducted where immunized splenocytes from DSG3−/−mouse or DSG3+/− mouse were transferred into RAG2−/− immunodeficiencymouse. RAG2−/− mouse expresses Dsg3 protein but has neither mature Tcells nor B cells because neither T cell receptor genes norimmunoglobulin genes can be rearranged in the mouse. Therefore it isassumed that the transferred splenocytes are not rejected and anti-Dsg3IgG can be produced in the recipient mouse.

DSG3−/− mice and DSG3+/− mice were sensitized with 5 μg of purifiedmouse rDsg3 by using complete Freund's adjuvant (0 day). Then boosterimmunization was carried out with mouse rDsg3 using incomplete Freund'sadjuvant after 7 and 14 days. The production of antibody was confirmedon the 18^(th) day by ELISA in the same manner as in Example 2. Finally,booster immunization was carried out with mouse rDsg3 but without anyadjuvant, and the mice were sacrificed several days after the boosterimmunization to prepare splenocytes as immune cells.

To perform the adoptive transfer of splenocytes, monocytes were isolatedfrom the spleens of DSG3−/− mice or DSG3+/− mice and re-suspended incomplete RPMI1640 medium (Nissui Pharmaceuticals, Tokyo) containing 10%fetal bovine serum, 0.21% sodium bicarbonate solution (w/v), 2 mML-glutamine (GIBCO), and antibiotics. About 1×10⁷ splenocytes weresuspended in PBS and transferred into RAG2−/− mouse via caudal vein byintravenous injection. The production of antibody was tested by ELISAusing mouse rDsg3 as a coating antigen in the same manner as in Example2.

Anti-Dsg3 IgG was detected in the blood of recipient RAG2−/− mice asearly as 4^(th) day after the transfer of DSG3−/− splenocytes. Theantibody produced rapidly increased and reached a plateau around the21^(st) day; the production then continued permanently (n=13) (FIG. 2A).The sustained antibody production was observed for 6 months or moreuntil the mice died. In contrast, anti-Dsg3 IgG was always undetectableover time in the blood of RAG2−/− mice in which DSG3+/− mousesplenocytes had been transferred (n=5) (FIG. 2A).

In order to determine the localization of B cells producing anti-Dsg3IgG, ELISPOT assay was conducted as follows. A 96-well microtiter plateof which bottom is made of PVDF (Millipore-Amicon, Beverly, Mass.) wascoated with mouse rDsg3 of 30 μg/ml. The monocytes prepared formreconstructed RAG2−/− mouse peripheral blood, spleen, bone marrow, andlymph node were incubated on the plate at 37° C. under humid aircontaining 5% Co₂ for 4 hours. The IgG bound to the membrane wasvisualized as a spot by using alkaline phosphatase-conjugated anti-mouseIgG antibody (Zymed Laboratories Inc, San Francisco, Calif.). The numberof spots were counted under a stereoscopic microscope, the frequency ofB cells producing anti-mDsg3 IgG was determined as the number per 10⁵monocytes. All the experiments were performed in triplicate. The numberof B cells producing anti-Dsg3 IgG determined by the assay is shown inTable 2.

It was revealed that B cells producing anti-Dsg3 IgG were localized inthe spleens and lymph nodes of recipient RAG2−/− mice in the early phase(on the 22^(nd) day) as well as late phase (on the 117^(th) day) afterthe adoptive transfer (Table 2). In this Table, RAG2−/− mice in whichsplenocytes from immunized DSG3−/− mouse had been transferred isrepresented by “+”; the mouse which had no transferred cell is by “−”(a). “Days” represents days from the transfer to the sacrifice (b). Thenumber of B cells producing anti-mDsg3 IgG is indicated as the numberper 10⁵ monocytes (c). The frequency of B cells producing anti-Dsg3 IgGin the spleen ranged from 20 to 100 cells per 10⁵ monocytes. TABLE 2Mouse Transfer^(a) Days^(b) Spleen Lymph node Bone marrow PBMC RAG#466−− −−    0.0 ± 0.0^(c)  0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 RAG#514 + 22  86.5± 29.9  13.5 ± 13.6 0.0 ± 0.0 3.8 ± 5.4 RAG#212 + 33 102.1 ± 14.7 47.8 ±8.8 0.0 ± 0.0 0.0 ± 0.0 RAG#134 + 117 20.8 ± 5.9 16.5 ± 5.9 2.1 ± 2.90.0 ± 0.0 RAG#135 + 117 31.3 ± 8.8 27.1 ± 2.9 0.0 ± 0.0 0.0 ± 0.0

EXAMPLE 5 RAG2−/− Mouse Having Immunized DSG3−/− Splenocytes Expressedthe Phenotype of Pemphigus Vulgaris

The first recognized symptom in recipient RAG2−/− mouse having immunizedDSG3−/− splenocytes was weight loss (n=13) as compared with mouse havingDSG3+/− splenocytes (n=5) around 7 to 14 days after the adoptivetransfer (FIGS. 2B and 3A). The weights of these mice then continued todecrease and several of them actually died. The remaining survived micelater began to gain their weights (FIG. 2B). The phenotype of weightloss was recognized in all the recipient RAG2−/− mice examined (n=13).Several of the recipient RAG2−/− mice (n=5) had the onset of erosionwith scab on the skin around the noses which is a common area of scratch(FIG. 3B).

In vivo IgG deposition was found on the cell surface of stratifiedsquamous epithelium keratinocyte including epidermis (FIG. 3G; aroundthe nose), mucous membrane of oral cavity (FIG. 3F, hard palate), andesophagus mucous membrane in recipient RAG2−/− mouse. In epidermisconsisting of several layers of keratinocytes, the presence of IgGdeposition was restricted to the lower layers (FIG. 3G), while IgG wasobserved in all the epithelial layers in epithelia of oral cavity andesophagus (FIG. 3F). IgG deposition was not detected in any othertissues including heart, lung, liver, kidney, stomach, small intestine,and large intestine in these mice. Histological diagnosis of RAG2−/−mice having immunized DSG3−/− splenocytes showed the presence ofintraepithelial cleavage immediately above the basal layer, namely,acantholysis immediately above the basal layer which is a typicalcharacteristic of pemphigus vulgaris, in buccal mucous membrane, hardpalate (FIG. 3C), the oral and pharyngeal region, and upper part of theesophagus (FIG. 3D). The significant inflammatory cell infiltrate wasnot essentially seen in blistering lesions in the early phase (FIG. 3C).Inflammatory infiltrate was chiefly found in old erosion foci (FIG. 3E).Irritation and acute inflammation secondarily caused by food wererecognized there, which were due to loss of epithelial barrier function.It can be presumed that these damages perhaps reduced ingestion in themice and resulted in growth inhibition.

In contrast, no phenotypic or pathological alterations were recognizedin RAG2−/− mice having immunized DSG3+/− splenocytes. These findingssuggested that RAG2−/− mice having immunized DSG3−/− splenocytesexpressed pemphigus vulgaris phenotype.

EXAMPLE 6 RAG2−/− Mouse Having Immunized DSG3−/− Splenocytes Exhibitedthe Phenotype of Telogen Hair Loss

About 15 to 25 days after the adoptive transfer, partial hair loss wasrecognized in 11 of 13 RAG2−/− mice (FIG. 2A, see arrow, FIG. 4A, B).Typically, hair loss was initiated as a small spot, and then wasexpanding gradually during the next 2 to 3 weeks. Hair loss wasinitiated in the forehead in recipient RAG2−/− mice of 12-week old orless, and it further expanded backward. A mosaic of new hair wasrecovered in the telogen hair loss spot in several mice, but there weremice in which telogen hair loss spots remained without changing for onemonth or more or in which telogen hair loss expanded without formingdemarcated telogen hair loss spot (FIG. 4D). When adhesive tape wasadhered to the area adjacent to a telogen hair loss spot and thenremoved (hair-pull test) (Koch, P. J., et al., J. Cell Sci.111:2529-2537 (1998)), a bunch of hairs adhered on the tape (FIG. 4C).These phenotypes lasted for 6 months or more as far as anti-Dsg3 IgG waspresent in the blood.

The skin biopsy of RAG2−/− recipient mouse revealed intense IgGdeposition on the cell surface of keratinocytes around hair bulb (FIGS.4G, H). The intensity of IgG binding in hair follicle was much higherthan that in epidermis (FIG. 4G). Histological diagnosis of the skinshowed the presence of acantholysis between cells around hair bulb atthe resting stage and the basal layer of outer root sheath (FIGS. 4E, H;arrow). In telogen hair loss spots, there were empty expanded telogenhair follicles being consistent with telogen effluvium (FIG. 4F). Therewas no evident acantholysis in the surface layer of epidermis withoutdamage. No obvious infiltration of inflammatory cells was recognizedaround the hair follicles with acantholysis (FIGS. 4E, F).

In contrast, no telogen hair loss spots were always found in RAG2−/−mice in which DSG3+/− splenocytes had been transferred.

INDUSTRIAL APPLICABILITY

The development of this model provided a new direction for the study oftissue-specific autoimmune diseases (autoimmune diseases in which therelation between target antigen and toxic antibody or T cell has beenclarified). The model of the present invention is useful to elucidatecellular mechanisms underlying the production of antibody againstantigen protein for an autoimmune disease and induction of cytotoxic Tcell by particularly modifying lymphocytes before adoptive transfer.This model can also be a valuable tool for the development of newdisease-specific therapies. Because, in the pemphigus vulgaris modelanimal in accordance with the present invention, the major phenotypesare weight loss and reversible telogen hair loss, activity of thedisease can be monitored by observing the mice without sacrificing them.ELISA titer of blood anti-Dsg3 antibody is also an objective index forthe disease activity. Further, the phenotype remains expressed for 6months or more. Thus efficacy of each therapeutic method can readily andobjectively be evaluated. More importantly, the method of the presentinvention is applicable for the development of active disease mousemodels for other tissues specific autoimmune diseases in which targetantigens have been identified.

1. A method for identifying a mouse recipient that produces an antibodyagainst an antigen protein for an autoimmune disease and/or hasactivated T cells reactive to the antigen protein, said methodcomprising: transplanting immune cells from a donor to the recipient,wherein (i) the donor lacks a gene encoding the antigen protein, anddevelops functional immune cells, and (ii) the recipient is the samespecies as the donor, and has the same genetic background as the donoror is immunodeficient; and identifying whether the recipient has anantibody and/or activated T cells reactive to the antigen protein. 2.The method according to claim 1 further comprising immunizing the donorwith the antigen protein prior to transplanting immune cells to therecipient.
 3. The method according to claim 1, wherein the recipient isimmunodeficient.
 4. The method according to claim 3, wherein therecipient lacks a RAG2 gene.
 5. The method according to claim 1, whereinthe immune cells are splenocytes.
 6. The method according to claim 1,wherein the antigen protein is desmoglein
 3. 7. The method according toclaim 1, wherein the autoimmune disease is pemphigus vulgaris.
 8. Amouse recipient that has been identified by the method according toclaim 6, wherein (i) the recipient has immune cells from a mouse donorthat lacks a gene encoding desmoglein 3 and develops functional immunecells, (ii) the recipient has the same genetic background as the donoror is immunodeficient, and (iii) the recipient produces an antibodyagainst desmoglein 3 and/or has activated T cells reactive to desmoglein3.
 9. The recipient according to claim 8, wherein the recipient isimmunodeficient.
 10. The recipient according to claim 9, wherein therecipient lacks a RAG2 gene.
 11. The recipient according to claim 8,wherein the recipient is suitable as a model of pemphigus vulgaris. 12.A method for determining efficacy of a test agent as a therapeutic foran autoimmune disease, said method comprising: treating a mouserecipient according to claim 8 with a test agent, and observing aphenotype of the mouse recipient, thereby determining the efficacy ofthe test agent.
 13. The method according to claim 12, wherein therecipient is immunodeficient.
 14. The method according to claim 13,wherein the recipient lacks a RAG2 gene.
 15. The method according toclaim 12, wherein the autoimmune disease is pemphigus vulgaris.